Pharmacology: Cell Culture, Fura-2 Assay and Flow Cytometry

Fura-2 assay on CHO cells. The fura-2 assay was performed as previously described³⁰ using a LS50 B spectrofluorimeter from Perkin Elmer (Überlingen, Germany).

Flow cytometric competition binding assay. The binding assay was performed as described elsewhere²⁹ with the following adaptations. Samples were measured without further processing using a FACSCalibur^TM flow cytometer from Becton Dickinson (Heidelberg, Germany), equipped with an argon laser (488 nm) and a red diode laser (635 nm); instrument settings were: FSC: E-1, SSC: 350 V, FL4: 800 V, Flow: HI; measurement stopped when 10000 gated events were counted. The experiments were performed using 490 µL of cell suspension, 5 µL of Cy5-pNPY (final concentration 5 nM) and 5 µL of test compound (100-fold concentrated). If indicated, 5 µL of Dy-635-pNPY (final concentration 10 nM) was used as fluorescent ligand.

Incubation time was 90 min at room temperature. Ki values were obtained from 2-3 independent experiments.

Flow cytometric selectivity assay. The selectivity of the new Y2R antagonists for human NPY Y2 over Y1, Y4 and Y5 receptors was proven by flow cytometric binding assays on Cy5-pNPY (Y1R, Y5R), and Cy5-[K⁴]-hPP (Y4R) according to previously described methods.^{29, 44-45} All flow cytometric measurements were performed on a FACSCalibur^TM flow cytometer from Beckton Dickinson (Heidelberg, Germany), equipped with an argon laser (488 nm) and a red diode laser (635 nm). The compounds were tested at two concentrations (1 µM and 10 µM) in duplicates.

3.6 References

1. Doods, H.; Gaida, W.; Wieland, H. A.; Dollinger, H.; Schnorrenberg, G.; Esser, F.;

Engel, W.; Eberlein, W.; Rudolf, K. BIIE0246: a selective and high affinity neuropeptide Y Y(2) receptor antagonist. Eur. J. Pharmacol. 1999, 384, R3-5.

2. Dumont, Y.; Cadieux, A.; Doods, H.; Pheng, L. H.; Abounader, R.; Hamel, E.; Jacques, D.; Regoli, D.; Quirion, R. BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist. Br. J. Pharmacol. 2000, 129, 1075-88.

3. Smith-White, M. A.; Hardy, T. A.; Brock, J. A.; Potter, E. K. Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions. Br. J. Pharmacol. 2001, 132, 861-8.

4. Dautzenberg, F. M.; Neysari, S. Irreversible binding kinetics of neuropeptide Y ligands to Y2 but not to Y1 and Y5 receptors. Pharmacology 2005, 75, 21-9.

5. Rudolf, K.; Eberlein, W.; Engel, W.; Mihm, G.; Doods, H. N.; Willim, K. D.; Krause, J.;

Wieland, H. A.; Schnorrenberg, G.; Esser, F.; Dollinger, H. Preparation of piperazine-containing peptidomimetics for use as NPY antagonists. DE 19816929, 1999.

6. Esser, F.; Schnorrenberg, G.; Dollinger, H.; Gaida, W. Preparation of novel peptides for use as NPY antagonists. DE19816932, 1999.

7. Dollinger, H.; Esser, F.; Mihm, G.; Rudolf, K.; Schnorrenberg, G.; Gaida, W.; Doods, H.

N. Preparation of novel peptides for use as NPY antagonists. DE 19816929, 1999.

8. Brennauer, A. Acylguanidines as bioisosteric groups in argininamide-type neuropeptide Y Y1 and Y2 receptor antagonists: synthesis, stability and pharmacological activity.

Doctoral thesis, Universität Regensburg, Regensburg, 2006.

9. Merten, N.; Lindner, D.; Rabe, N.; Rompler, H.; Mörl, K.; Schöneberg, T.; Beck-Sickinger, A. G. Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J. Biol. Chem. 2007, 282, 7543-51.

10. Salaneck, E.; Holmberg, S. K.; Berglund, M. M.; Boswell, T.; Larhammar, D. Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y2(1). FEBS Lett. 2000, 484, 229-34.

11. Berglund, M. M.; Fredriksson, R.; Salaneck, E.; Larhammar, D. Reciprocal mutations of neuropeptide Y receptor Y2 in human and chicken identify amino acids important for antagonist binding. FEBS Lett. 2002, 518, 5-9.

12. Akerberg, H.; Fallmar, H.; Sjodin, P.; Boukharta, L.; Gutierrez-de-Teran, H.; Lundell, I.;

Mohell, N.; Larhammar, D. Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions. Regul. Pept. 2010, 163, 120-9.

13. Fallmar, H.; Kerberg, H.; Gutierrez-de-Teran, H.; Lundell, I.; Mohell, N.; Larhammar, D.

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes. Neuropeptides 2011.

14. Kanno, T.; Kanatani, A.; Keen, S. L.; Arai-Otsuki, S.; Haga, Y.; Iwama, T.; Ishihara, A.;

Sakuraba, A.; Iwaasa, H.; Hirose, M.; Morishima, H.; Fukami, T.; Ihara, M. Different binding sites for the neuropeptide Y Y1 antagonists 1229U91 and J-104870 on human Y1 receptors. Peptides 2001, 22, 405-13.

15. Sautel, M.; Rudolf, K.; Wittneben, H.; Herzog, H.; Martinez, R.; Munoz, M.; Eberlein, W.; Engel, W.; Walker, P.; Beck-Sickinger, A. G. Neuropeptide Y and the nonpeptide antagonist BIBP 3226 share an overlapping binding site at the human Y1 receptor. Mol.

Pharmacol. 1996, 50, 285-92.

16. Kraus, A.; Ghorai, P.; Birnkammer, T.; Schnell, D.; Elz, S.; Seifert, R.; Dove, S.;

Bernhardt, G.; Buschauer, A. N(G)-acylated aminothiazolylpropylguanidines as potent and selective histamine H(2) receptor agonists. ChemMedChem 2009, 4, 232-40.

17. Xie, S. X.; Ghorai, P.; Ye, Q. Z.; Buschauer, A.; Seifert, R. Probing ligand-specific histamine H1- and H2-receptor conformations with NG-acylated Imidazolylpropylguanidines. J. Pharmacol. Exp. Ther. 2006, 317, 139-46.

18. Igel, P.; Schneider, E.; Schnell, D.; Elz, S.; Seifert, R.; Buschauer, A. N(G)-acylated imidazolylpropylguanidines as potent histamine H4 receptor agonists: selectivity by variation of the N(G)-substituent. J. Med. Chem. 2009, 52, 2623-7.

19. Keller, M.; Bernhardt, G.; Buschauer, A. [3H]UR-MK136: a highly potent and selective radioligand for neuropeptide Y Y1 receptors. ChemMedChem 2011, 6, 1566-71.

20. Keller, M.; Erdmann, D.; Pop, N.; Pluym, N.; Teng, S.; Bernhardt, G.; Buschauer, A.

Red-fluorescent argininamide-type NPY Y(1) receptor antagonists as pharmacological tools. Bioorg. Med. Chem. 2011, 19, 2859-78.

21. Keller, M.; Pop, N.; Hutzler, C.; Beck-Sickinger, A. G.; Bernhardt, G.; Buschauer, A.

Guanidine-acylguanidine bioisosteric approach in the design of radioligands: synthesis of a tritium-labeled N(G)-propionylargininamide ([3H]-UR-MK114) as a highly potent and selective neuropeptide Y Y1 receptor antagonist. J. Med. Chem. 2008, 51, 8168-72.

22. Dains, F. B.; Wertheim, E. The action of ammonia and amines on the substituted ureas and urethanes. II. Allophanic ester. J. Am. Chem. Soc. 1920, 42, 2303-2309.

23. Altmann, E.; Aichholz, R.; Betschart, C.; Buhl, T.; Green, J.; Lattmann, R.; Missbach, M. Dipeptide nitrile inhibitors of cathepsin K. Bioorg. Med. Chem. Lett. 2006, 16, 2549-54.

24. Jacobi, P. A.; DeSimone, R. W.; Ghosh, I.; Guo, J.; Leung, S. H.; Pippin, D. New syntheses of the C,D-ring pyrromethenones of phytochrome and phycocyanin. J. Org.

Chem. 2000, 65, 8478-89.

25. Weiss, S.; Keller, M.; Bernhardt, G.; Buschauer, A.; König, B. N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity. Bioorg. Med. Chem. 2010, 18, 6292-304.

26. Fujii, N.; Otaka, A.; Ikemura, O.; Akaji, K.; Funakoshi, S.; Hayashi, Y.; Kuroda, Y.;

Yajima, H. Trimethylsilyl Trifluoromethanesulfonate as a Useful Deprotecting Reagent in Both Solution and Solid-Phase Peptide Syntheses. J. Chem. Soc., Chem. Commun.

1987, 274-275.

27. Igel, P.; Schnell, D.; Bernhardt, G.; Seifert, R.; Buschauer, A. Tritium-labeled N(1)-[3-(1H-imidazol-4-yl)propyl]-N(2)-propionylguanidine ([(3)H]UR-PI294), a high-affinity histamine H(3) and H(4) receptor radioligand. ChemMedChem 2009, 4, 225-31.

28. Brennauer, A.; Keller, M.; Freund, M.; Bernhardt, G.; Buschauer, A. Decomposition of 1-(w-aminoalkanoyl)guanidines under alkaline conditions. Tetrahedron Lett. 2007, 48, 6996-9.

29. Ziemek, R.; Brennauer, A.; Schneider, E.; Cabrele, C.; Beck-Sickinger, A. G.;

Bernhardt, G.; Buschauer, A. Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands. Eur. J.

Pharmacol. 2006, 551, 10-8.

30. Müller, M.; Knieps, S.; Gessele, K.; Dove, S.; Bernhardt, G.; Buschauer, A. Synthesis and neuropeptide Y Y1 receptor antagonistic activity of N,N-disubstituted w-guanidino- and w-aminoalkanoic acid amides. Arch. Pharm. 1997, 330, 333-42.

31. Keller, M.; Teng, S.; Bernhardt, G.; Buschauer, A. Bivalent argininamide-type neuropeptide y y(1) antagonists do not support the hypothesis of receptor dimerisation.

ChemMedChem 2009, 4, 1733-45.

32. Velazquez-Campoy, A.; Todd, M. J.; Freire, E. HIV-1 protease inhibitors: enthalpic versus entropic optimization of the binding affinity. Biochemistry 2000, 39, 2201-7.

33. Ghorai, P.; Kraus, A.; Keller, M.; Gotte, C.; Igel, P.; Schneider, E.; Schnell, D.;

Bernhardt, G.; Dove, S.; Zabel, M.; Elz, S.; Seifert, R.; Buschauer, A. Acylguanidines as bioisosteres of guanidines: NG-acylated imidazolylpropylguanidines, a new class of histamine H2 receptor agonists. J. Med. Chem. 2008, 51, 7193-204.

34. Bodanszky, M.; Ondetti, M. A.; Birkhimer, C. A.; Thomas, P. L. Synthesis of arginine-containing peptides through their omithine analogs. Synthesis of arginine vasopressin, arginine vasotocin, and L-histidyl-L-phenylalanyl-L-arginyl-L-tryptoph-ylglycine. J. Am.

Chem. Soc. 1964, 86, 4452-9.

35. Leschke, C.; Storm, R.; BreitwegLehmann, E.; Exner, T.; Nurnberg, B.; Schunack, W.

Alkyl-substituted amino acid amides and analogous di- and triamines: New non-peptide G protein activators. J. Med. Chem. 1997, 40, 3130-3139.

36. Waring, W. S.; Whittle, B. A. Basic dihydromorphanthridinones with anticonvulsant activity. J. Pharm. Pharmacol. 1969, 21, 520-30.

37. Tadros, Z.; Lagriffoul, P. H.; Mion, L.; Taillades, J.; Commeyras, A. Enantioselective Hydration of Alpha-Aminonitriles Using Chiral Carbonyl Catalysts. J. Chem. Soc., Chem. Commun. 1991, 1373-1375.

38. Moriguchi, T.; Yanagi, T.; Kunimori, M.; Wada, T.; Sekine, M. Synthesis and properties of aminoacylamido-AMP: chemical optimization for the construction of an N-acyl phosphoramidate linkage. J. Org. Chem. 2000, 65, 8229-38.

39. Moura, C.; Vitor, R. F.; Maria, L.; Paulo, A.; Santos, I. C.; Santos, I. Rhenium(V) oxocomplexes with novel pyrazolyl-based N4- and N3S-donor chelators. Dalton Trans.

2006, 5630-40.

40. Bergeron, R. J.; McManis, J. S. Reagents for the Stepwise Functionalization of Spermine J. Org. Chem. 1988, 53, 3108-3111.

41. Imming, P.; Yang, X.-G. On the reaction of dicarboxylic anhydrides with 1,w-diamines.

Arch. Pharm. (Weinheim, Ger.) 1994, 327, 747-750.

42. Quelever, G.; Kachidian, P.; Melon, C.; Garino, C.; Laras, Y.; Pietrancosta, N.; Sheha, M.; Louis Kraus, J. Enhanced delivery of gamma-secretase inhibitor DAPT into the brain via an ascorbic acid mediated strategy. Org. Biomol. Chem. 2005, 3, 2450-7.

43. Gers, T.; Kunce, D.; Markowski, P.; Izdebski, J. Reagents for efficient conversion of amines to protected guanidines. Synthesis 2004, 1, 37-42.

44. Schneider, E.; Mayer, M.; Ziemek, R.; Li, L.; Hutzler, C.; Bernhardt, G.; Buschauer, A.

A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes. Chembiochem 2006, 7, 1400-9.

45. Ziemek, R.; Schneider, E.; Kraus, A.; Cabrele, C.; Beck-Sickinger, A. G.; Bernhardt, G.;

Buschauer, A. Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence. J. Recept.

Signal Transduct. Res. 2007, 27, 217-33.

Receptor Antagonists

4.1 Introduction

For many years G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomers. However, there is a growing number of reports that suggest homo- and hetero-dimerization of numerous GPCRs, e.g. adrenergic receptors,¹ GABAB receptors,^2-3 opioid receptors,^4-6 muscarinic receptors,^7-9 vasopressin receptors,^10-11 chemokine receptors,^12-13 dopamine receptors,^14-15 histamine receptors,^16-17 etc.^18-21 FRET experiments clearly proved the ability of human neuropeptide Y receptors Y1, Y2 and Y5 (hY1R, hY2R, hY5R) to form homo-dimers, whereas the hY2R is less prone to dimerization than the hY1R and the hY5R, respectively.²² More detailed investigations on the localization, formation and composition of Y2R dimers were performed by Parker et al. on different tissues of the rabbit.^23-24 Therein, the Y2R was observed to be largely dimeric in the kidney, but monomeric in the forebrain, possibly due to variable levels of G-proteins containing the Gαi subunit in different tissues.²⁴ Furthermore, in the renal cortex a protein-complex of about 300 kDa was detected, which was postulated to result from the association of both Y2R protomers of the homo-dimer with two G-protein αβγ hetero-trimers.²³ The dimerization might be enabled by a tryptophan-tryptophan H-bonding in TM4,²⁴ as suggested for dopamine D2 receptor dimers.²⁵ Commonly applied techniques for the identification of GPCR oligomers include co-immuno-precipitation,²⁶ functional studies (e.g. measurement of GPCR-β-arrestin interactions with β-galactosidase enzyme complementation technique²⁷), mutation studies of putative dimerization motives,²⁸ bioluminescence resonance energy transfer (BRET)¹ and molecular modeling.²⁹ However, all experiments demonstrating a physical interaction between GPCRs have up to now been performed ex vivo in transfected cells, which implies non-physiological cellular environments and – in most cases – unrealistically high concentrations of receptors in the membranes. In this context, the in vivo demonstration of GPCR dimers of the luteinizing hormone receptor (LHR) by Rivero-Müller et al. constitutes a major contribution to the proof of GPCR oligo-merization.³⁰ Co-transfection with a binding-deficient and a signaling-deficient LHR resulted in a quasi wild-type phenotype after activation with LH. Thus, functional activity could only be generated by the dimerization of the loss-of-function LHR mutants.³¹

The use of bivalent ligands as an alternative to the aforementioned methods offers the possibility to investigate native GPCRs, expressed endogenously by normal wild-type cells. Such compounds, defined as two pharmacophoric moieties linked through a spacer, should bring more insight into the phenomenon of GPCR dimerization.

Moreover, the bivalent ligand approach is discussed as an alternative drug design strategy. On the one hand, bivalent ligands can interact with the orthosteric and an additional allosteric binding site (cf. Figure 4.1). Such key interactions may be formed with residues that reside within extracellular loop domains,³³ which often display lower homology between receptor subtypes in comparison to transmembrane domains. Thus, a higher degree of receptor-subtype selectivity may be achieved by this approach. On the other hand, bivalent ligands addressing two protomers should enhance tissue specificity, as receptor oligomers (especially hetero-oligomers) are co-expressed in certain tissues with a distinct pharmacology and function.²⁷ Hence, concerning side-effects, ligands combining two distinct pharmacophoric moieties in one molecule should be superior to combined administration of two monomeric drugs. This renders the bivalent ligand approach a very interesting topic in drug design.

However, there are a few spatial requirements to be fulfilled for a successful application of bivalent ligands: firstly, the spacer length must be sufficient to address the binding sites of both protomers. Secondly, flexibility and appropriate chemical nature of the linker are essential for the correct positioning of each pharmacophore.

Studies on bivalent based drug design applied to opioid and serotonin receptors suggested a distance between the binding sites of 22-32 Å (corresponding to 16-24 atoms).^{32, 34-35}

The distance between the two binding sites is difficult to predict as there are several variants of contact dimers discussed in the literature.^{25, 32, 34} Molecular modeling stu-dies revealed a distance of about 27 Å in

case of a TM5,6 interface, while a TM4,5 interface leads to a substantially longer distance of approximately 32 Å.³² Also, the linker might span the transmembrane do-mains or it may bridge the two binding sites on the extracellular side, complicating the prediction of an optimal spacer length.

Moreover, an affinity-enhancing effect might occur due to cooperativity by inter-action with an allosteric site, rather than bridging the orthosteric pockets of two protomers (Figure 4.1). Thus, length and chemical nature of the spacer must be optimized for each GPCR dimer empirically

Figure 4.1. Illustration of the bivalent ligand concept for bridging of a hypothetical GPCR dimer. a) Occu-pation of an allosteric binding site by the second pharmacophore of a bivalent ligand. b) The unoc-cupied dimer undergoes univalent binding followed by either bridging of the bivalent ligand, or binding of a second ligand to give the dimer with both sides occupied. Adopted from Portoghese.³²

bivalent ligand a)

b)

by screening several ligands.³⁶ To date, besides the aforementioned opioid and serotonin receptors, a variety of successful applications of the bivalent ligand approach to GPCRs is reported in the literature.^36-39 In case of NPY receptors, the first studies focused on the preparation of peptidic bivalent antagonists derived from porcine NPY (pNPY). The first dimeric ligands, consisting of two nonapeptides mimicking the C-terminal part, were bridged by either disulfide bonds, 2,6-diaminopimelic acid and 2,3-diaminopropionic acid, respectively. These antagonists showed high affinity for both the Y1R and the Y2R (Ki (Y1R) = 0.2-2.9 nM; Ki (Y2R) = 0.02-8.8 nM).⁴⁰ Balasubramaniam et al. constructed the first highly potent and selective Y1R bivalent antagonist based on the C-terminal hexapeptide of NPY.⁴¹ The only non-peptidic bivalent ligands for the Y1R known so far were prepared in our laboratory as analogs of BIBP 3226⁴² linked by a spacer attached to the guanidino group.⁴³

This chapter presents the first non-peptide bivalent Y2R antagonists, derived from BIIE 0246⁴⁴ by application of the guanidine-acylguanidine bioisosteric approach in analogy to the recently developed Y1R bivalent ligands. The amine precursors used for the linkage with dicarboxylic acids are presented in Figure 4.2.

Figure 4.2. Argininamide-type amine precursors derived from BIIE 0246 used for the preparation of bivalent Y2R antagonists.

4.2 Chemistry

The synthesis of the amine precursors ((S)-3.48-(S)-3.50, (S)-3.52 and (S)-3.54), which were used for the preparation of bivalent Y2R antagonists is described in chapter 3 (for structures cf. Figure 4.2). These building blocks contain two amino groups prone to acylation, namely the free N^ω´H2 and the terminal amine in the acyl- or carbamoyl linker. The terminal amino function is more reactive than the acylguanidine due to higher basicity and less sterical hindrance. Hence, the guanidino function was not protected as the synthetic strategy aimed at a minimum number of reaction steps and maximal purity on the low mg scale rather than at optimization of yields and synthetic routes. Various alkanedioic acids and amine precursors, respectively, were employed to synthesize bivalent ligands with spacer lengths between 16 and 35 atoms linking the two pharmacophoric moieties. Furthermore, the aforementioned building blocks (Figure 4.2) were chosen for linkage in order to obtain compounds differing in the chemical nature of the spacer.

For the preparation of the bivalent ligands 4.1-4.9 the amine precursors were acylated with the corresponding alkanedioic acid in an EDC/DMAP-mediated coupling step under microwave irradiation followed by purification with preparative HPLC (Scheme 4.1; for structures of the bivalent ligands 4.1-4.11 cf. Table 4.1). In case of the antagonists 4.10 and 4.11, the terminal amino group of the branched linker was Boc-protected to avoid side reactions. Thus, acidic deprotection was necessary prior to the purification of the product (Scheme 4.1). The branched linkers were kindly provided by Dr. M. Keller from our laboratory.⁴³

Scheme 4.1. Synthesis of the bivalent ligands 4.1-4.11. Reagents and conditions: a) alkanedioic acid (1 eq), amine precursor (2.4 eq), EDC × HCl (2.1 eq), NEt3 (6 eq), DMAP (cat.), CH2Cl2, DMF, MW, 35 min, 65 °C; b) TFA/CH2Cl2 1:1 (v/v), 2 h, rt.

The title compounds were obtained in low yields (10-30 %). HPLC reaction monitoring revealed four major peaks (Figure 4.3 shows a representative chromatogram for the synthesis of 4.6). After HPLC purification several compounds were identified by MS analysis. Peak A corresponds to non-reacted amine precursor which was used in excess. Peak B is assigned to a trifluoroacetylated by-product. This side reaction resulted from the application of the amine precursors as TFA salts. As these salts proved to be highly stable, they were not converted into the less stable free amines before coupling (see also Chapter 3.2.2). The monoacylated compound corresponds to peak C, whereas peak D with the longest retention time was identified as the respective bivalent ligand.

Figure 4.3. HPLC reaction monitoring of the preparation of bivalent ligands exemplified by the synthesis of 4.6.

Conditions: eluent: mixtures of acetonitrile + 0.025 % TFA (A) and 0.025 % aq. TFA (B), gradient: 0 to 30 min: A/B 20/80 to 95/5, 30 to 40 min: 95/5. Chromatogram cut after 30 min as there were no further peaks. Peak A: starting material (S)-3.50 (tR = 17.2 min), peak B: TFA-acylated compound (tR = 18.6 min), peak C: monoacylated by-product (tR = 19.8 min), peak D: product 4.6 (tR = 19.8 min).

4.3 Pharmacological Results and Discussion

The synthesized bivalent ligands were investigated for Y2R binding in a flow cytometric binding assay using CHO cells, stably expressing the hY2R,⁴⁵ and fluorescence-labeled Cy5-pNPY. NPY Y2R antagonistic activities were determined in a spectrofluorimetric Ca²⁺ assay (fura-2 assay) on CHO cells, stably expressing the hY2R.⁴⁶ The selectivity for the Y2R over Y1R, Y4R and Y5R was examined by means of flow cytometry.⁴⁷

0 5 10 15 20 25 30

0.0 0.2 0.4 0.6

A

D = 4.6 B

C

time / min

Signal / AU

Table 4.1. Structures, Y2R antagonistic activities (KB) and binding affinities (Ki) of the bivalent Y2R antagonists.

a Number of atoms between the two guanidine functions (“length” of the spacer). ^b Inhibition of 70 nM pNPY-induced [Ca²⁺]i mobilization in CHO cells; mean values ± SEM (n = 2-3). ^c Flow cytometric binding assay using Cy5-pNPY (KD = 5.2

Figure 4.4. Displacement of 5 nM Cy5-pNPY by (S)-3.47 (BIIE 0246) and the acyl-guanidine-type bivalent ligands 4.1, 4.2, 4.6, 4.10, respectively (mean values ± SEM; n = 2).

Functional and binding data as well as the structures of the prepared bivalent compounds are summarized in Table 4.1. All bivalent NPY Y2R antagonists showed binding affinities in the two to three-digit nM range. In most cases, the KB values determined in the calcium assay were even lower. Rigidization by introduction of a double-bond in the spacer is moderately tolerated by the receptor as shown for compound 4.1 in comparison with the flexible analog 4.2 (Ki (4.1) = 161 nM vs. Ki (4.2)

= 69 nM; Figure 4.4). The acylguanidine-bridged ligands 4.2-4.5 showed the highest affinities with linker lengths of 22 and 30 atoms, respectively, bridging the two pharmacophoric moieties (Ki (4.2) = 61 nM; Ki (4.5) = 69 nM), whereas Y2R anta-gonistic activities decreased with increasing spacer lengths. The introduction of a second positively charged amino group in the linker of antagonist 4.6 led to a gain in affinity. With a Ki value of 21 nM 4.6 (spacer length: 30 atoms) was the most potent bivalent ligand of this small series (Figure 4.4), confirming a possible additional electrostatic interaction with the receptor, as already discussed for the monovalent ligands (cf. Chapter 3).

The affinity of carbamoylguanidine 4.9 (spacer length: 20 atoms) corresponds to about half of that of the lower homologs 4.7 and 4.8 with spacer lengths of 16 and 18 atoms, respectively. The Ki values of the latter are in the same range of those of 4.2 and 4.5. Unfortunately, acylguanidine analogs of 4.7-4.9 have not been available, yet, in order to compare the impact of alkanoyl- and carbamoyl-linkage on binding. As already observed for argininamide-type Y2R antagonists (cf. Chapter 3), the presence of the carbamoyl NH-group might implicate a different orientation of the N^G -substituent. Moreover, carbamoylguanidines are slightly more basic than acyl-guanidines. This should be affinity-enhancing, as the guanidino group is supposed to undergo an ionic interaction with an acidic residue of the Y2R.

All bivalent Y2R ligands show mono-phasic competition binding curves with Hill slopes not statistically significantly different from unity. Furthermore, the binding affinities of the bivalent antagonists are lower than that of the parent compound BIIE 0246.

Thus, bridging of a Y2R dimer, or an allosteric modulation by these bivalent ligands is

-10 -9 -8 -7 -6 -5

very unlikely. Nevertheless, a larger library of structurally diverse bivalent Y2R ligands is necessary for more detailed structure-activity investigations.

The bivalent ligands 4.10 and 4.11 with a terminal positively charged amino function in the branched part of the linker revealed Ki values below 100 nM. These compounds are considered appropriate precursors for the synthesis of radio- or fluorescence-labeled bivalent Y2R antagonists.

The introduction of a second pharmacophoric entity into the bivalent ligands might change the selectivity profile, as observed for several argininamide-type bivalent Y1R antagonists. A few eutomeric (R,R)-configured compounds appeared to be highly potent ligands⁴³ with a decreased selectivity against Y4R, and some of the distomeric (S,S)-configured antagonists⁴³ turned out to bind at the Y4R, rather than at the Y1R.⁴⁸ Thus, the high selectivity of monovalent antagonists used as building blocks is not necessarily retained with the corresponding bivalent ligands. Nevertheless, in the case of the bivalent Y2R antagonists presented in this thesis, the Y2R selectivity was not compromised compared to the monovalent parent compounds, as confirmed by flow cytometric binding studies (Table 4.2).

Table 4.2. NPY receptor subtype selectivity of the bivalent Y2R antagonists.

Table 4.2. NPY receptor subtype selectivity of the bivalent Y2R antagonists.

Im Dokument Application of the Guanidine–Acylguanidine Bioisosteric Approach to NPY Y2 (Seite 119-0)